Patient-derived parathyroid organoids as tracer and drug-screening application model

Parathyroid diseases are characterized by dysregulation of calcium homeostasis and alterations in parathyroid hormone (PTH) excretion. The understanding of parathyroid hyperplastic growth and the development of parathyroid-targeted treatment and imaging tracers could benefit from in vitro models. Therefore, we aim to establish stem cell-derived, three-dimensional organoids representing human parathyroid tissue in vitro. Patient-derived hyperplastic parathyroid tissue was dispersed and parathyroid organoids (PTO) were cultured and characterized. PTO-derived cells were shown to exhibit in vitro self-renewal over several passages, indicative of the presence of putative stem cells. Immunofluorescence and RNA-sequencing confirm that PTO phenocopy hyperplastic parathyroid tissue. Exposure of PTO to increasing calcium concentrations and to PTH-lowering drugs resulted in a significantly reduced PTH excretion. Next to this, the PTO showed specific binding of 11C-methionine to the targeted receptor. Additionally, when organoids were incubated with 99mTc-sestamibi, we observed a higher uptake in PTOs from patients with a 99mTc-sestamibi positive scan compared to patients with a negative scan. These data show functionality of PTOs resembling the parathyroid. In conclusion, we present a patient-derived PTO culture, that recapitulates the originating tissue on gene and protein expression and functionality. This PTO model paves the way for future physiology studies and therapeutic target and tracer discovery.


Introduction
The parathyroid glands usually are four delicate structures of 3-4 mm in size, situated close to the thyroid gland, and functionally maintaining calcium homeostasis by secreting parathyroid hormone (PTH).
Calcium is an essential element for the nervous, muscular and skeletal systems. Parathyroid glands consist of chief-and oxyphil cells, the former of which express calcium-sensing receptor (CaSR), and regulate the blood calcium concentration by mediating PTH secretion (1). Oxyphil cells are less prevalent than chief cells and increase in number with age (2). Parathyroid diseases are heterogeneous conditions that generally are caused by alterations in PTH secretion, resulting in dysregulation of calcium homeostasis. In primary hyperparathyroidism, a common parathyroid disease, reduced CaSR expression has been noted (3)(4)(5), resulting in uncontrolled continuous release of PTH by parathyroid hyperplasia and leading to subsequent hypercalcemia (6).
The understanding of parathyroid hyperplastic growth and the development of effective parathyroid tracers for diagnostic imaging and drugs for targeted treatment could benefit from in vitro models derived from human hyperplastic parathyroid tissue. To develop a model for studying the functional and proliferative properties of the parathyroid gland, two rat-derived parathyroid clonal epithelial cell lines were previously developed illustrating in vitro calcium-dependent PTH responses (7,8). Only one of these cell lines expressed a wide range of parathyroid-related genes (7). In further attempts to develop an in vitro parathyroid model, bovine parathyroid glands were cultured that showed long-term serial propagation of parathyroid cells (9)(10)(11)(12). However, these cultures were swiftly overgrown by fibroblastic cells and rapidly lost calcium responsiveness (11,12). Besides the use of animal derived parathyroid cells, human parathyroid cell cultures have been investigated. These cultures had a lowproliferative activity and were readily terminally differentiated in a 2D environment (13)(14)(15)(16), making them less useful for an in vitro model.
Organoids are 3D structures that closely recapitulate tissue architecture and cellular composition and are developed from stem cells (17). Organoid models have been developed from multiple exocrine and endocrine glandular tissues (18)(19)(20)(21)(22)(23)(24)(25)(26). These models have proven very useful for studying tumor behaviour and assessing drug responses, and have provided a platform for long-term in vitro experimentation (18)(19)(20)(21)(22)(23). Hence, this study aimed to isolate human parathyroid stem cells from primary tissue and study their potential to expand and form parathyroid organoids (PTO) in vitro that recapitulate functional parathyroid tissue. In addition, we assess the potential of such a patient-derived PTO model to perform physiology studies and the ability to be used for the development of potential novel therapeutic targets, drug screening and imaging tracer testing.

In vitro self-renewal and organoid formation from human parathyroid stem cells
In total, fifty hyperplastic parathyroid gland biopsies were dissociated into single cells and cell clumps using mechanical and enzymatic digestion. These cells were cultured in parathyroid medium, and resulted in the formation of parathyroid spheres ( Figure 1A-B, passage 0). To determine the presence of potential stem cells in parathyroid tissue, the in vitro self-renewal potential of cells derived from parathyrospheres was assessed. Seventeen of 50 cultures were used to determine organoid forming efficiency, with 100% of cultures reaching passage 1 (p1), 41% reaching p2, 35% reaching p3 and 18% reaching p4 with a maximum organoid-forming efficiency (OFE) of 6.6% in p1, 2.7% in p2 and 2.9% in p3 ( Figure 1B-C). Thus, self-renewal of parathyroid cells was shown. The parathyroid organoids (PTOs) continued to secrete PTH during culture ( Figure 1D). To further characterize PTOs, gene expression of parathyroid markers was evaluated by quantitative Polymerase Chain Reaction (qPCR). Geneexpression analysis of parathyroid markers PTH, CaSR, and GATA Binding Protein 3 (GATA3), a transcription factor involved in the development of parathyroid tissue and in adult parathyroid cell proliferation (27), showed a stable expression between passages 1-3 (no significant differences in expression between passages, all p-values > 0.065) (Supplemental Figure 1).

Figure 1.
In vitro self-renewal of organoid-forming cells from human putative parathyroid stem cells. A. Schematic representation of parathyroid tissue isolation, primary tissue culture, and the selfrenewal assay. B. Primary parathyroid spheres seven days in culture (passage 0), and PTOs at the end of passage 1, 2 and 3. Scale bar = 100 µm C. Self-renewal potential of PTOs during passaging (n=19 patients error bars represent range). D. Secreted PTH levels from PTOs during passaging (n=4 patients, error bars represent SEM). PTO= parathyroid organoid.

Characterization of parathyroid organoids
To evaluate whether the putatative parathyroid stem cells are able to differentiate into parathyroid gland specific cells, the cells were subjected to a modified Bingham protocol (28,29) involving continuously exposure to 100 ng/mL Activin, 100 ng/mL Sonic hedgehog (Shh) and 5% fetal bovine serum (FBS) for 21 days with media changes every seven days. Previous studies showed that the Bingham protocol successfully replicated parathyroid differentiation using human embryonic stem cells or tonsil-derived mesenchymal stem cells. These cells induced a significant release of PTH with response to increasing or decreasing calcium levels (28)(29)(30).
During differentiation, the cells increased slightly in size, however no further morphological changes were observed ( Figure 2B). PTH secretion increased from the first week of differentiation until the second week ( Figure 2C). Only in week 2 of differentiation, the PTH levels differed significantly from the PTH levels secreted prior to differentiation (average increase in PTH of 46.2% ± 21.6% in week 1 (p=0.166) and of 58.6% ± 2.6% in week 2 (p=0.002)). To further characterize PTOs and differentiated PTOs (dPTO), gene expression levels were assessed using bulk RNA-sequencing to compare hyperplastic parathyroid tissue (n=2), PTOs (n=3) and Bingham Protocol dPTOs (n=3). When assessing the number of differentially expressed genes (DEGs) between hyperplastic parathyroid tissue and PTOs, we only observed 25 up-, and 61 downregulated genes out of 14,900 evaluated genes. Moreover, between tissue and dPTOs, we observed 50 up-, and 82 down-regulated genes out of 14,900 evaluated genes. Only two out of 35 genes of the parathyroid markers ( Figure 3) were differentially expressed in both PTOs and dPTOs compared to tissue. These two genes were PTH and CHGA, both excreted factors, and both downregulated in PTO and dPTOs compared to tissue. Thus, similarity in gene expression between tissue and PTOs and dPTOs was observed, especially in parathyroid-specific genes ( Figure 3). However, we did observe inter-patient variability in the gene expression of tissue (i.e. between tissue of patient 1 and 2), which was also reflected in the respective PTOs and dPTOs (Supplemental Figure 2, Supplemental Table 1).
When comparing tissue, PTOs and dPTOs of one single patient (e.g. of patient 1), limited variability was observed (Supplemental Figure 2).
Genes that were differentially expressed in tissue compared to PTOs and dPTOs (Supplemental  Tables 4-7).
Interestingly, genes such as STC1 (Stanniocalcin 1) and CCL2 (C-C Motif Chemokine Ligand 2) that were significantly upregulated in both PTO and dPTO compared to tissue were amongst others associated with calcium ion transport. (Supplemental Tables 4 and 6). Genes significantly upregulated in the dPTO group compared to tissue seem to involved in the regulation of cell population proliferation (Supplemental Table 6). These organoids were treated following the modified Bingham differentiation protocol, which might be the reason of this possibly more proliferative state, since in PTOs this GO term was not detected (Supplemental Tables 4 and 6). Furthermore, genes significantly upregulated in PTO compared to tissue were associated with angiogenesis and vascularization. No parathyroid-specific biological processes, such as hormone production and calcium ion transport, were associated with downregulated in both PTO and dPTO conditions when compared to tissue (Supplemental Figure 5, Supplemental Tables 5 and 7).
Moreover, we found that all parathyroid-specific genes (31-34) expressed in hyperplastic parathyroid tissue samples were also expressed in PTOs and dPTOs ( Figure 3A). Glial Cells Missing Transcription Factor 2 (GCM2), a zinc finger-type transcription factor, is known to be a master regulator for embryonic development of parathyroid glands (35). Since these parathyroid glands are not formed in GCM-null mice, GCM2 is believed to be essential for development of the parathyroid glands and survival at the earliest stage after organ specification during the embryogenesis (35)(36)(37)(38). Chromogranin A (CHGA) is an acidic protein in secretory granules of (neuro)endocrine cells and is a major protein of the parathyroid gland that is co-stored and co-secreted with PTH (39,40). When we focus on four parathyroid specific genes, i.e., GCM2, CHGA, PTH and CASR, gene expression of all was observed, albeit to a lower extend, in dPTO and PTO compared to primary tissue ( Figure 3B). Immunolabeling of these important parathyroid markers was confirmed at the protein level ( Figure 4). When comparing immunolabeling of parathyroid tissue with parathyroid organoids, similarities were seen in the expression pattern of GCM2, CHGA, PTH and CaSR ( Figure 4).  and SOX2, when comparing PTOs and dPTOs to tissue (Supplemental Figure 6). Additionally, none of the stem cell markers (Supplemental Figure 6) were differentially expressed in both PTOs and dPTOs compared to tissue.

Functionality of parathyroid organoids
PTH secretion is known to be negatively regulated by increased extracellular calcium levels in normal physiology. Therefore, we examined whether the release of PTH by PTO was responsive to extracellular calcium ( Figure 5A). We observed a significant decrease of 31.3% ± 10.8% (p=0.015) when organoids were subjected to very high levels of calcium (3.4 mM), when compared to normal culturing conditions (=1.4 mM) ( Figure 5B). When the organoids were then switched back to basal 1.4 mM calcium, we observed a significant increase of 53.0% ± 11.1.% (p=0.018) ( Figure 5B). These results indicate the fully functional response of PTOs.

Drug screening
To demonstrate the utility of patient-derived PTO in testing potential novel therapeutic targets, validated therapeutics were used to assess the effect of the drug on PTH secretion by the parathyroid organoids ( Figure 6A). Cinacalcet HCl (cinacalcet), an allosteric modulator of CaSR that increases the sensitivity of the CaSR to activation by extracellular calcium, suppresses PTH secretion in patients with primary hyperparathyroidism or secondary hyperparathyroidism on maintenance dialysis (41). Calcitriol, a vitamin D analogue, inhibits the production of PTH by acting directly on the parathyroid gland vitamin D receptor, thereby inhibiting transcription of the gene encoding PTH for the synthesis of PTH and indirectly by increasing calcium uptake in the gastrointestinal system, maintaining PTH secretion at low levels (42,43). We observed a significant decrease in PTH secretion when organoids were subjected to different concentrations of cinacalcet compared to DMSO control (average reduction of 58.9% ±8.5% for 1.5 µM and 58.8% ± 12.9% for 2.5 µM, p=0.020 and p=0.045) ( Figure 6B). A similar effect was observed when organoids were exposed to different concentration of calcitriol (average reduction of 50.3% ± 3.3% for 2 nM and 54.4% ± 2.0% for 20 nM, p<0.0001 and p<0.0001, respectively). parathyroid tissue isolation, primary tissue culture, and the drug-screening experiment. B. Secreted PTH levels from PTOs exposed to 1.5µM and 2.5µM cinacalcet (n=3 patients, error bars represent the SEM, normalized to PTH levels in the DMSO control = dotted line). C. Secreted PTH levels from PTOs exposed to 2nM and 20nM calcitriol (n=3 patients, error bars represent the SEM, normalized to PTH levels in the DMSO control = dotted line). ***= significant (p<0.05), PTO= parathyroid organoid.

PET/SPECT tracer validation
To demonstrate the utility of patient-derived PTOs to validate PET/SPECT tracers, validated tracers, i.e. 11 C-methionine (44-47) and 99m Tc-sestamibi (48)(49)(50), were used to assess uptake of the tracer by the PTOs. 11 C-methionine is an amino acid tracer used to localize parathyroid adenomas using a PET (positron emission tomography) scan. The 11 C-methionine uptake mechanism is not yet fully understood; it is presumed to be involved in the synthesis of pre-pro-PTH, a PTH precursor which results in intense and specific uptake in hyperfunctioning parathyroid glands (51,52). Also, the exact mechanism of 99m Tcsestamibi uptake in parathyroid glands remains unknown. 99m Tc-sestamibi uptake depends on numerous factors, including perfusion, cell cycle phase and functional activity (53). It has been suggested that the electrical potential of the plasma and mitochondrial membrane regulates uptake of 99m Tc-sestamibi and that tissues rich in mitochondria are avid for it (54,55). 99m Tc-sestamibi is detected using a gamma-camera which can acquire SPECT (single photon emission computed tomography) 3D images.

Discussion
In this paper, we report the establishment of a patient-derived parathyroid organoid culture, showing self-renewal potential suggesting the presence of stem cells. Furthermore, we have shown that these PTOs resemble the originating tissue on both gene and protein expression levels and functionality. We illustrate clinically relevant responses (increased and decreased hormone secretion) of these PTOs in response to changes in calcium concentration and PTH lowering drugs. Furthermore, we also found specific parathyroid-targeted tracer uptake in our PTOs, all together demonstrating that these organoids can model human parathyroid functionality.
We confirm that our organoids phenocopy primary parathyroid tissue using RNA-sequencing.
In fact, the observed heterogeneity in gene expression between patients seemed to reflect in the respective organoids. The GO biological process of "regulation of cell population proliferation" was significantly upregulated in dPTOs compared to tissue, in contrast to PTOs, suggesting that this upregulation is the result of exposing PTOs to the modified Bingham differentiation protocol.
Furthermore, when organoids were exposed to this differentiation protocol for two weeks, it is possible to increase the PTH excretion with an additional 50%.
Regretfully, we were unable to find evidence for the existence of unique parathyroid stem cells in our in vitro PTO model. This is in line with results from a recent study by van der Vaart et al. in thyroid organoids who could not find evidence for the existence of unique thyroid stem cells (26). They described that many solid tissues do not rely on a professional stem cell for their maintenance and repair, but rather temporarily recruit differentiated cells into a transient progenitor pool (26), suggesting that this phenomenon is also applicable to the thyroid or in this case even parathyroid gland.
For the clinical development of new imaging tracers and drugs, an established appropriate preclinical model for testing is required (56). We used our culturing method to culture parathyroid organoids to perform functional testing. Manipulating the calcium concentration and using clinically available PTHlowering drugs, resulted in a significantly reduced PTH excretion of PTOs, demonstrating that these organoids can model human parathyroid functionality and are a valid in vitro model. This effect is similar to the response expected in patients. Next to this, we demonstrated that the PTOs showed specific binding of the PET tracer 11 C-methionine and SPECT tracer 99m Tc-sestamibi. This indicates that we may be able to culture patient-specific PTOs. This methodology could also be used to predict patient response to treatment using our organoid model and develop or test new effective parathyroid imaging tracers.
This study is the first to develop a patient-derived PTO culture methodology of parathyroid tissue. Previous studies aimed at culturing bovine or rat parathyroid glands in 2D (7)(8)(9)(10)(11)(12). However, those cell culture models of bovine origin fail due to fibroblast cell overgrowth, and rapid loss of calcium responsiveness (10)(11)(12). Since cells in vivo experience a complex 3D environment that exposes them to circulating molecules, neighbouring cells, and the extracellular matrix, 2D cell cultures are unable to mimic the complicated microenvironment cells experience in tissue (57). Organoids are self-organizing, 3D culture systems that are highly similar to actual human organs, including increased exposure to circulating molecules, neighbouring cells, and the extracellular matrix (Matrigel) (18,(58)(59)(60)(61)(62). Organoids from several glandular tissues have been developed, such as thyroid organoids (24), papillary thyroid cancer organoids (63), salivary glands (64), and prostate cancer organoids (65). The analysis of organoid formation could provide valuable insight about the mechanisms underlying human development and organ regeneration (58). Such analysis highlight the value of organoids for discovering potential novel therapeutic targets, opening up opportunities for future imaging tracer testing and targeted treatment screening. We encountered a similar effect in our earlier organoid cultures but lowering the fibroblast growth factor 10 (FGF10) concentration solved this problem. We did see a small increase in fibroblasts in later passages (p3). However, these organoids are significantly larger then in earlier passages (p1/p2), and sometimes lack sufficient support of the Matrigel. Thereby exposing the PTO to the topography of the culture plate potentially leading to epithelial -mesenchymal transition (66).
The limitations of our study include the absence of the original microenvironment. This consists of, amongst others, fluctuating concentrations of extracellular signals and the presence of blood vessels.
This may be the reason why our PTOs do not have exactly the same transcriptome as hyperplastic parathyroid tissue. The limited differences that were observed, likely do not affect the functionality of our PTOs, since the functional testing and tracer experiment all show that the PTOs are a highly suitable model that resembles functional parathyroid tissue. Furthermore, it has been shown that microenvironmental factors are able to alter the therapy response of cells, complicating the use of organoids for these studies (67,68). Additional research is needed to further explore the clinical application of this parathyroid culture methodology as one day these parathyroid organoids may aid as a stem cell transplantation method for patients with abnormally low levels of PTH (hypoparathyroidism).
For this application, first a comparison has to be made between healthy parathyroid tissue and hyperplastic parathyroid tissue.
In conclusion, we present a patient-derived parathyroid organoid culture that recapitulates the originating tissue on gene and protein expression and functionality. This parathyroid culture will be able to provide a novel in vitro model to perform physiology studies and discover potential novel therapeutic targets, opening opportunities for future tracer testing and drug screening of the parathyroid gland.

Patient material
Human benign hyperplastic parathyroid tissue was obtained from patients undergoing parathyroid surgery. The biopsies were transported from the operating room (OR) in Hank's Balanced Salt Solution (HBSS) (Gibco, NY, USA) with 1% bovine serum albumin (BSA) (Gibco), on ice for immediate further processing.

Self-renewal
To study the long-term self-renewing potential, organoids were dissociated and re-plated for the next passage. Prior to passaging, Matrigel was dissolved by incubation with Dispase enzyme (1 mg/mL for 30 minutes at 37 °C; Sigma) and organoids with a size >50 µm were counted using the Ocular Micrometer at a 10x objective. Initial parathyrosphere cultures of seven days old (d) were dissociated to single cells using 0.05% trypsin-EDTA (Invitrogen), counted using Tryphan blue and cell concentration was adjusted to 2 x 10 6 cells per ml. In total, 20 μL of this cell solution was combined on ice with 40 µL of Basement Membrane Matrigel (BD Biosciences) and plated in the center of a 24-well tissue culture plate. After solidifying the Matrigel for 30 minutes at 37 °C, gels were covered in 500 µL of parathyroid medium as defined above and parathyroid medium was renewed weekly. This self-renewal procedure was repeated every three to four weeks and up to 5 times (4 passages).

Organoid differentiation
PTOs were encapsulated in Matrigel as described above and subjected to a modified Bingham Protocol for differentiation (28,29). Standard culture medium, DMEM:F12 containing Pen/Strep antibiotics (Invitrogen), and Glutamax (Invitrogen). In short, cells were continuously exposed to 100 ng/mL Activin A (Immunotools), 100 ng/mL Shh (R&D systems) and 5% FBS for 14 days with media changes every seven days.

PTH secretion
Medium was collected before passage and before each time point during the differentiation period, and stored at -20 °C until use. Medium was used to measure the concentration of secreted PTH protein by the PTOs. Secreted PTH was measured by electro-chemical luminescence immuno assay (ECLIA) using an Elecsys PTH kit (Roche, Germany).

Effect of extracellular calcium on PTH secretion
Undifferentiated organoids were exposed to parathyroid medium containing normal (

Drug screening
Cinacalcet HCL (Combi-Blocks, QA-8513) was added to the medium in two different concentrations (1.5 µM and 2.5 µM) and following 72 hours of incubation, the medium was collected to measure the concentration of secreted PTH protein by the PTOs. The concentration of Cinacalcet HCL used was based on literature that investigated the effect on parathyroid cultured cells (74,75). Further, calcitriol (Cayman Chemical, 32222-06-3) was added to the medium in two concentrations (2 nM and 20 nM) and following 72 hours of incubation, the medium was collected to measure the concentration of secreted PTH protein by the PTOs. The concentration of calcitriol used was also based on a previous study (76).
Both cinacalcet and calcitriol were dissolved using DMSO, therefore DMSO was used as vehicle control in a concentration of 0.1%.

PET/SPECT tracer validation
To demonstrate the utility of patient-derived PTOs to validate PET/SPECT tracers, existing tracers, i.e. 11 C-methionine and 99m Tc-sestamibi, were used to assess uptake of the tracer by the PTOs. Tracer uptake was corrected for the number of organoids by counting the number of organoids organoids using the Ocular Micrometer at a 10x objective prior to the experiment. Uptake was expressed as percentage of organoid-associated radioactivity per 10,000 organoids. Uptake values were corrected for background radiation. All experiments were executed in duplicate or triplicate, depending on the number of organoids.

Data availability.
The raw bulk RNA-Seq data (fastq files) used in this study have been deposited in the NCBI's Gene Expression Omnibus (GEO) (accession number GSE197445).

Statistics
All data represent the mean ± SEM. Statistical analyses and producing graphs were done using GraphPad Prism, version 8 (GraphPad Software). The statistical methods relevant to each figure are described in the figure legends. Statistical comparisons between two groups were performed using the Mann-Whitney U test or Student's t test (depending on normal distribution). P values of less than 0.05 were considered to indicate a significant difference between groups.

Study approval
Written informed consent of participants was obtained prior to participation. The study was approved by the Medical Ethics Committee Groningen (approval no. 2017/640). All authors have read and agree to the published version of the manuscript.

Acknowledgements
We would like to thank the surgeons and pathology departments of the University Medical Center Groningen, Treant location Bethesda, and the Martini Hospital for collecting and distributing hyperplastic parathyroid gland tissue. Sequencing was performed with the excellent assistance from Diana Spierings and the ERIBA Research Sequencing Facility. Part of the work was performed at the UMCG Imaging and Microscopy Center (UMIC).